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<OL> <LI> Profile the in vivo whole blood RNA and cytokine response to Salmonella Typhimurium (ST) infection in animals with high and low fecal shedding phenotypes. <LI> Using the same animals in Objective 1, profile the in vitro whole blood RNA and cytokine response to exposure to the general inflammatory endotoxin, lipopolysaccaride. <LI>Annotate response profiles for common expression patterns and functional themes and develop regulatory network information on response to inflammatory stimuli. <LI> Develop predictive tools for identifying pigs with a low fecal shedding phenotype in naive pigs and in pigs post-infection. </ol>
We predict that through the successful completion of these objectives, we will identify important genes and pathways that are relevant to understanding what aspects of porcine immune responses that are effective in decreasing shedding and/or leading to improved food safety. Such information will also be used to develop a classification tool to predict the important phenotype of shedding of bacterial pathogens associated with animal and pork contamination. If such a predictor can be validated using experimental and field populations, this approach can then be tested for heritability and utility in genetic improvement to potentially decrease Salmonella shedding and pork contamination.
NON-TECHNICAL SUMMARY: The long-term goal of this project is to improve food safety by decreasing fecal shedding of Salmonella bacteria on the farm and transmission to the food chain. Our central hypothesis is that the use of functional genomics will identify genes and immune networks that control desirable production phenotypes such as reduced pathogen shedding and/or induction of protective immunity post-infection. <P> The immediate goals of this project are to: a) create a new challenge population of 100 animals to be used to determine the genes and pathways in whole blood that respond in vivo following Salmonella infection, analyzing changes in gene expression profiles in whole blood samples and correlating with shedding over 21 days post-inoculation; b) determine the in vitro response to Salmonella lipopolysaccaride (LPS) in whole blood from these animals during shedding episodes, in recovering pigs, and in na?ve pigs; c) analyze this new data, as well as our substantial prior data on porcine response to Salmonella, to understand the networks and pathways underlying these host responses; d) further develop a classification tool from these data to predict shedding phenotypes; and e) validate this classifier by taking advantage of the fact that two collaborators are already collecting Salmonella shedding data on 7,200 pigs. We propose to collect blood samples on a subset of their population and validate the prediction classifier on selected shedder and non-shedder animals (a case-control study).
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APPROACH: <BR>
Disease challenge with Salmonella typhimurium (ST) RNA profiling: We will infect 100 pigs with ST, and quantify fecal shedding at 0, 1, 2, 7, 14, and 21 dpi. Blood on all animals will be collected at 0, 2 and 21 days post infection for RNA isolation and ELISA assay of serum cytokines, and EDTA tubes for CBC. Samples from the top 10% of animals (lowest level of shedding from 7-21 dpi) and the bottom 10% will be processed for Affymetrix analysis. Differentially expressed genes will be identified by a repeated-measures analysis of these normalized data, to identify gene sets that respond to infection and that differ in expression across pigs with different levels of shedding and/or effective immune response.
<P>ELISA and QPCR analysis of Cytokines and their RNAs: We will measure six inflammatory pathway cytokines (IFNG, IL1B, IL6, IL8, IL10, and TNF) serum protein and blood RNA on all animals. Cytokine/RNA levels will be compared to shedding levels to look for correlation, and these data will also be used to confirm that differences in gene expression patterns seen are due to response to ST. Blood will also be collected in EDTA tubes for complete blood counts. RNA profiling- in vitro analyses- and statistics: Blood will be incubated at 37 degrees C +/- LPS for 6 hrs. Post-treatment, blood will be processed for hybridization, data collection, analysis and cytokine measurement as above.<P>
Bioinformatics: We will use ANEXdb to store the expression data and to create export files for data submission to NCBI-GEO and to further analyze this data. We will use publicly available RNA profiling and cytokine data from pig, human and mouse in vitro and in vivo studies and from the above work to develop an understanding of genes and pathways responding to ST infection or LPS treatment. In vivo expression profiles can be compared across different stages/times of infection and to naive animals, and comparisons can be made between in vivo expression profiles and in vitro response profiles at similar and different infection times. Finally, in vitro responses of whole blood samples from naive animals will be compared to responses from similarly stimulated blood from day 2 infected animals, thus exploring whether there is a difference in the in vitro response between a naive animal and an infected animal. <P>Classifier testing and development: We will test the initial classifier(s) on samples collected above. We will also test the classifier(s) on RNA isolated from ~500 blood RNA samples collected on the farms as part of the OConnor/McKean project. Once Salmonella qualitative shedding data is available, we will process all Sal+ samples (~25-30) and an equal number of Sal- samples. These RNAs will be used in QPCR analysis using both the set of cytokine genes and the preliminary classifier to determine the efficacy of the classifier to predict the shedder phenotype in animals for which prior history is unavailable. We will also use all Objective 1 and 2 Affymetrix data to create and evaluate several new classifiers using Weka software.